This invention relates generally to the field of apoptosis, and more specifically to the inhibition of Fas-mediated apoptosis.
Necrosis and apoptosis are two basic processes by which cells may die. In necrosis, cell death usually is a result of cell injury. The cells tend to swell and lyse, and the cell contents ultimately spill into the extracellular space. By contrast, apoptosis is a mode of cell death in which single cells are deleted in the midst of living tissues. Apoptosis, or programmed cell death, is utilized in a number of biological processes that include modeling the embryo, regulating the immune system, and tumor regression (M. D. Jacobson et al., Cell, 347, 1997; A. Winoto, Curr. Opin. Immunol., 9, 365, 1997; G. Evan, et al., Science, 281, 1998; H. Arai et al., Proc. Natl. Acad. Sci. USA, 94, 13862, 1997). For example, apoptosis accounts for most of the programmed cell death in tissue remodeling and for the cell loss that accompanies atrophy of adult tissues following withdrawal of endocrine and other growth stimuli. In addition, apoptosis is believed to be responsible for the physiologic death of cells in the course of normal tissue turnover (i.e., tissue homeostasis) (Kerr, J. F.,et al, 1972. Br. J. Cancer 26:239-257; Wyllie, A. H., et al. 1980. Int. Rev. Cytol. 68:251-306). As an example, apoptosis is observed in the immune system as the process by which B and T lymphocytes are removed when they fail to recognize a foreign antigen or when they are self-reactive.
The deregulation of programmed cell death may result in a disease state. Dysfunction of the apoptotic system has been implicated in oncogenesis, development of autoimmunity and degenerative diseases. Degenerative diseases which result from excessive cell death include degenerative neurological diseases, such as Alzheimer""s disease and Parkinson""s disease which are associated with the death of particular subsets of neurons. The inopportune death of T cells in AIDS may be associated with physiological cell death. Physiological cell death may also be associated with transplant rejection. Diseases due to increased cellular proliferation are also possible due to deregulation of the apoptotic mechanism and include autoimmune diseases in which self-reactive B and T cells are allowed to persist. The term xe2x80x9cphysiological cell deathxe2x80x9d is used here to describe cell death that occurs by a mechanism that exists in the mammal to kill its own cells and includes apoptosis and programmed death as synonymous terms.
An imbalance of the cell proliferation and cell degeneration processes also may lead to development of neoplasias in cells deregulated for the control of apoptosis. As a protective mechanism against cancer, tumor necrosis factor can trigger apoptosis in transformed host cells. An important example of the type of cancers which develop when cell proliferation exceeds the normal balance, is human follicular lymphoma. As with other malignancies where the development of neoplasia is related to an oncogene, follicular lymphoma is characterized by a chromosomal breakpoint. The rearrangement in follicular lymphoma is the most common chromosomal translocation in human lymphoid malignancies, the t(14;18)(q32;q21) translocation, which is known to inhibit programmed cell death in B cells. The bcl-2 gene is translocated and deregulated in follicular lymphoma.
Intense studies of apoptosis in the last decade have identified membrane bound receptors, and their cognate ligands that together begin a program that ultimately leads to cell death (A. Ashkenazi, Science, 281, 1305, 1998). One of the most characterized receptors is Fas (also called CD95 or Apo-1) belonging to the tumor necrosis factor receptor (TNFR) superfamily (N. Itoh et al., Cell, 66, 233 1991; A. Oehm et al., J. Biol. Chem., 267, 10709, 1992). Binding of soluble or cell-surface expressed CD95 ligand to CD95 leads to oligomerization of the receptor and the subsequent the transmission of the apoptosis signal. Fas has three cysteinexe2x80x94rich extracellular domains and an intracellular death domain (DD) required for signaling (N. Itoh et al., J. Biol. Chem., 268, 10932, 1993). Ligation of the receptor by its cognate ligand, FasL (T. Suda et al., Cell, 75, 1169, 1993), or an agonistic antibody (S. Yonehara et al., J. Exp. Med., 169, 1747, 1989), leads to the recruitment of a cytoplasmic adapter molecule FADD (also called MORT-1), mediated by a DD in FADD and the DD of Fas (A. M. Chinnaiyan et al., Cell, 81, 505, 1995; Boldin et al., J. Biol. Chem., 270, 7795, 1995). Additionally, FADD contains a death effector domain (DED) that recruits the protease caspase-8 (also called FLICE, MACH and Mch5) to the signaling complex M. Muzio et al., Cell, 85, 817, 1996; M. P. Boldin et al., Cell, 85, 803, 1996; S. M. Srinivasula et al., Proc. Natl. Acad. Sci. USA, 93, 14486, 1996). This zymogen, through proximity with other caspase-8 molecules, is cleaved rendering it fully active, thus beginning a protease cascade that leads to cell death (M. Muzio et al., J. Biol. Chem., 273, 2926, 1998). A counterpoint to this activation is inhibition of apoptosis.
Apoptosis plays an important role in the homeostasis and development of all tissues within an organism. The present invention relates to a novel apoptotic-associated polypeptide Lifeguard (LFG), and to the use of LFG antibodies, nucleic acid sequences, and amino acid sequences in the study, and treatment of apoptosis-associated disorders.
A substantially purified lifeguard (LFG) polypeptide is provided. The polypeptide includes an amino acid sequence as set forth in SEQ ID NO:2, or a conservative variant thereof.
An isolated polynucleotide is provided that encodes an amino acid sequence as set forth in SEQ ID NO:2, or a conservative variant thereof. An isolated polynucleotide is provided selected from the group consisting of SEQ ID NO:1, where T can also be a U, SEQ ID NO:1, a nucleic acid sequence complementary to SEQ ID NO:1, and fragments of SEQ ID NO:1 that are at least 15 bases in length and that hybridize under highly stringent conditions to DNA which encodes a polypeptide as set forth in SEQ ID NO:2.
An antibody is provided that binds to a polypeptide having an amino acid sequence as set forth in SEQ ID NO:2.
A method is provided for identifying a compound which affects a function a polypeptide as set forth in SEQ ID NO:2, or a conservative variant thereof, or affects the expression of a polynucleotide including a sequence as set forth in SEQ ID NO:1. The method includes incubating the compound and a cell expressing the polypeptide under conditions sufficient to allow the compound to interact with the cell, determining the effect of the compound on Fas-mediated cell death, and comparing Fas-mediated cell death of the cell contacted with the compound with Fas-mediated cell death of a cell not contacted with the compound.
A method is provided for treating a subject with a disorder associated with decreased Fas-mediated cell death, including administering to the subject a therapeutically effective amount of a compound that inhibits LFG function or expression. A method is also provided for treating a subject with a disorder associated with increased Fas-mediated cell, including administering to the subject a therapeutically effective of a compound that augments LFG function or expression.
A method is provided for treating a patient having or at risk of having a disorder associated with increased Fas-mediated cell death, including introducing into a cell of a patient having a disorder associated with Fas-mediated cell death a polynucleotide sequence encoding a SEQ ID NO:2 operatively linked to a promoter, thereby inhibiting Fas-mediated cell death.
A pharmaceutical composition is provided that includes a therapeutically effective amount of a substantially pure LFG polypeptide as set forth as SEQ ID NO:2, or a conservative variant thereof, and a pharmaceutically acceptable carrier.
A kit for is provided for detecting the presence of LFG in a sample. The kit includes a carrier means being compartmentalized to receive therein one or more containers comprising a container containing an antibody which specifically binds to LFG. A kit is also provided that is useful for the detection of a target LFG nucleic acid sequence. The kit includes a carrier means being compartmentalized to receive therein one or more containers including a container containing oligonucleotides which hybridize to LFG nucleic acid sequences.
A transgenic nonhuman animal is provided wherein the transgenic animal has a phenotype characterized by expression of LFG, otherwise not naturally occurring in the animal, the phenotype being conferred by a transgene contained in the somatic and germ cells of the animal. The transgene includes a nucleic acid sequence which encodes SEQ ID NO:2. In one embodiment, transgenic animals having gene knockouts in which the target gene has been rendered nonfunctional by an insertion targeted to the gene to be rendered non-functional by homologous recombination are provided.